It is evident from many different experiments, that the human nucleotide excision repair (NER) apparatus first distinguishes structural DNA perturbations caused by bulky lesions derived from the reactions of metabolically activated environmental carcinogens that enter the human body. This step entails the formation of XPC/HR23B complexes that partially open the duplex near the lesion site. The initial structural distortions caused by the lesions and the subsequent strand separation suggest that base-base stacking and hydrogen bonding interactions are first weakened and then broken during these initial phenomena. In this project, we will examine the effects of structurally different lesions of different adduct conformations on these initial structural distortions, and then use variations in base sequence context as a tool to modulate these base stacking interactions and thus gain insight into the specific structural factors that affect human NER activity. In Specific Aim 1, the Structural basis of recognition and processing of DNA damage by the human NER apparatus will be investigated. This aim will be focused on DNA lesions of different chemical structure, physical size, and conformational properties positioned at the same site in otherwise completely identical sequence contexts. In Specific Aim 2, the local DNA distortions caused by the lesions will be modulated by varying the base sequence context in which the lesions are embedded, and determine effects of different flanking bases on the structural characteristics and changes in NER activity. In Specific aim 3, the effects of adduct structure and base sequence on binding and patterns of helix opening by the human NER lesion-recognizing XPC/HR23B heterodimer duplex will be investigated. The results of this project will have ultimate translational identification by providing new information about DNA lesiosn that are resistant to DNA repair, thus providing important information about biomarkers of exposure of the human population to environmental carcinogens.